Summary of Work: The aim of this project is to determine the roles of two unique hsp70 heat-shock proteins in male germ cell development and function. The hsp70 proteins are molecular chaperones that assist in the folding of nascent polypeptides and in refolding of denatured proteins following heat shock and other stresses. HSP70-1 and -3 proteins are highly induced in most cells in response to stress but poorly induced in spermatogenic cells, while HSP70-2 and HSC70T proteins are expressed only in these cells and in response to developmental cues. Because HSP70-2 is synthesized in abundance during meiosis and is similar to other hsp70 proteins known to be chaperones, we hypothesized that HSP70-2 serves such a role for proteins involved in critical events during this phase of spermatogenesis. This was confirmed when a knock-out of the Hsp70-2 gene resulted in developmental arrest and apoptosis of all pachytene spermatocytes at the G2/M-phase transition of meiosis I. SInce this event requires cyclin B1-dependent Cdc2 kinase activity, the results suggested that HSP70-2 is a chaperone required for Cdc2 activation. Although Cdc2 was present in the testis of Hsp70-2 knock-out mice, it did not form a heterodimer with cyclin B1 and lacked kinase activity. Addition of recombinant HSP70-2 protein to a homogenate of testis from Hsp70-2 knock-out mice restored the ability of Cdc2 to form a heterodimer with cyclin B1 and to become an active kinase, confirming that HSP70-2 is a chaperone for Cdc2. The HSC70T protein is present only in spermatids, during the post-meiotic phase of male germ cell development. By analogy with HSC70-2, we hypothesized that HSC70T is a chaperone for unique proteins involved in post-meiotic germ cell development. However, male Hsc70t knock-out mice are fertile and sperm development is not altered. This suggests that other hsp70 proteins present in spermatids can compensate for the absence of HSC70T protein during sperm development under normal conditions. It remains to be determined if lack of HSC70T increases the sensitivity of spermatids or sperm to the effects of heat-shock or environmental chemicals.